Thrombosis within the cardiovascular system is believed to be the principal mechanism for vasoocclusive disorders, which are responsible for much morbidity and mortality in Western societies. Thus, antithrombotic drugs have been used extensively for treating many of these disorders (see, for example, Stein et al., Circulation (1989) 80:1501-1513). As with any group of drugs, however, none is entirely satisfactory for all individuals, and additions to the repertoire of possible therapeutic agents are always welcome.
The mechanism for the formation of thromboses is complex, but partially understood. An initial trauma, such as that initiated by rupture of atherosclerotic plaques or following mechanical removal of plaques during angioplasty, results in adhesion of platelets to the damaged vessel wall through platelet-to-nonplatelet interactions, and subsequent platelet aggregation (platelet-to-platelet interactions), in combination with deposition of fibrin. This sequence of events is controlled by the interaction of plasma proteins with specific platelet surface glycoprotein receptors. Since platelet adhesion is thought to be the initial response to injury, it is an especially desired target for inhibition in order to prevent or ameliorate thrombosis and/or restenosis that may be mediated by adherent platelets.
Unstimulated circulating platelets contain receptors for several adhesive proteins. Among these are laminin, which binds to VLA 2 and VLA 6, and collagen, which binds to VLA 2, GPIV, and others. The initial attachment of platelets to the subendothelium is believed to be mediated by the binding of the GPIb-IX complex residing on the platelet surface to von Willebrand Factor (vWF), which is immobilized in the vessel wall, particularly under the high shear rate conditions found at the sites of arteriovascular occlusion. The platelet GPIb-IX complex, while functional generally on resting platelets, normally does not bind plasma-borne vWF. Under normal circumstances, the arterial surface does not provide adhesive protein ligands (vWF) for platelet adherence; thus, platelet adhesion is limited to the vWF bound at the sites of vascular damage.
When the presence of bound vWF supports the adhesion of the platelets to the endothelium, the platelets become activated and are then capable of forming platelet aggregates, associated with the binding of fibrinogen (Fg) and plasma-borne vWF, through the nowactivated GPIIb-IIIa receptor. It follows that substances which specifically inhibit the adhesion of the unactivated platelets through the GPIb-IX-bound vWF interaction would interrupt the formation of thromboses, especially in vessels wherein high shear stress is caused by stenoses.
The GPIb-IX complex, formed from the Ib surface membrane heterodimer (Ib.sub..alpha. and Ib.sub..beta.) noncovalently complexed with GPIX, is present at a density of approximately 25,000 copies/platelet surface. The absence of this complex has been shown to be responsible for Bernard-Soulier Syndrome, a rare congenital bleeding disorder characterized by the absence of GPIb-IX complex displayed on the platelet surface and defective arterial adhesion of platelets. Defects in von Willebrand factor, such as those characterizing von Willebrand Disease also have been shown to lead to defective arterial adhesion of platelets.
Substances capable of interfering with GPIb-IX/vWF interaction are known. Kirby, Thrombon. Diathres. Haemorph (1975) 34:770, reports that Evans' blue dye inhibits the ristocetin-induced binding of vWF to formaldehyde-fixed platelets in vitro. Geratz et al, Thromb Haemostasis (1978), 39:411, showed the same effect by aromatic amidino compounds. Phillips et al, Blood (1988) 72:1898-1903, showed that ristocetin-induced platelet agglutination as well as shear-induced platelet aggregation in platelet-rich plasma was effectively inhibited by the triphenylmethyl compound aurin tricarboxylic acid (ATA) at concentrations tenfold lower than those of other compounds previously described. ATA has also been demonstrated to be an effective inhibitor of coronary artery thrombosis in vivo (Strony et al, Circulation (1989) 80:II-23 (Abstract); PCT Application WO 89/04166.
Binding of vWF to GPIb-IX complex has also been inhibited by monoclonal antibodies immunoreactive with the GPIb-IX complex as disclosed by Ruan et al, Brit J Haemotol (1981) 49:511; and Coller et al, Blood (1983), 61:99-119(1983). The antibodies inhibit ristocetin-induced binding of vWF to platelets. Becker et al, Blood (1989) 74:690-694, showed that one of these antibodies or it immunoreactive fragments block GPIb function in guinea pigs in vivo, although they exert no effect on platelet aggregation induced by ADP, collagen, or thrombin.
Monoclonal antibodies immunoreactive with human vWF block platelet adhesion to collagen at high shear rates (Fressinaud et al, J Lab Clin Med (1988) 112:58-67, and Cadroy et al., Circulation (1989) 80:Suppl. II-24). Murine monoclonal antibodies against porcine von Willebrand factor induce an antithrombotic state in normal pigs without effecting intrinsic platelet function (Bellinger et al., Proc Natl Acad Sci (USA) (1987) 84:8100-8104).
A proteolytic 45 kd fragment of glycocalicin (a GPIb fragment) and its derivatives inhibit binding of vWF to platelets, and these peptides can be used as antithrombotic agents, as shown in EPO Publication No. 317278. Fragments of vWF also inhibit this binding, as described in Australian Patent Application AU87/73715. Vicente et al., J Biol Chem (1990) 265:274-280, discloses peptides derived from GPIb which block ristocetin- and botrocetin-induced binding of vWF to platelets.
The peptides of the present invention provide alternative approaches to antithrombosis by specifically inhibiting the binding of vWF with the GPIb-IX platelet-borne complex. These are effective agents in antithrombotic therapies.